Mass screening and treatment (MSAT) for malaria elimination lacks an ideal diagnostic tool to allow sensitive and affordable test of the target population in the field. This study evaluated whether Capture and Ligation Probe-PCR (CLIP-PCR) could be used in a field MSAT in Laiza City, Myanmar.Plasmodium in a 20 m2 laboratory with workbench, a biosafety cabinet, a refrigerator, a benchtop shaking incubator and a qPCR machine, by four technicians using CLIP-PCR with sample pooling, at a health clinic of the Chinese bordering town of Nabang. On day 2, all positives were followed up and treated.On day 0, two dried blood spots were collected from each participant. On day 1, all samples were screened for Plasmodium vivax, Plasmodium falciparum, and P. vivax/P. falciparum mix, respectively. The testing capacity was 538 persons/day, with a cost of US$0.92 /person. The proportion of submicroscopic infection was 64.7%. All positive individuals received treatment within 72 h after blood collection.Of 15,038 persons  screened, 204 (1.36%) were CLIP-PCR positives. Among them, 188, 14, and 2 were infected with Using CLIP-PCR in MSAT in low transmission settings can support the malaria elimination efforts in the China-Myanmar border region.The online version contains supplementary material available at 10.1186/s12936-023-04449-x. Plasmodium vivax outbreaks could be dangerous because of a high rate of glucose-6-phosphate dehydrogenase deficiency (up to14.8%) in the population of the target district [Mass screening and treatment (MSAT) for malaria is defined as testing of an entire population in a wide geographical area followed by treating only positive individuals, regardless of whether they have malaria symptoms. Such an approach provides important information on the epidemiology of malaria, which can be useful for further disease containment efforts , and is district . Althougdistrict , 4.Plasmodium genus- and species-identification with a much-shortened assay time [To overcome this challenge, a high-throughput molecular assay on a 96-well plate platform called Capture and ligation probe-PCR (CLIP-PCR) has been developed , enablinP. vivax was observed in Laiza City and the surrounding areas in 2016 [Kachin Special Region II (KSR2), a poor mountainous area located in northern Myanmar, shares a land border of 214.6 km with Yingjiang County of Yunnan Province, China. Since the regional conflict in KSR2 in 2012, a large number of internally displaced persons immigrated and resettled along the China-Myanmar border, posing a risk to the achievements of joint cross-border malaria prevention and control activities between China (Yunnan Province) and Myanmar –14. In K in 2016  to 185 iP. vivax outbreak and reducing the cross-border malaria importation into Yunnan, China.In this study, the authors evaluated the feasibility of using CLIP-PCR in MSAT to determine the malaria prevalence in the Laiza district of Myanmar. Understanding the level of malaria prevalence in Laiza district can provide a basis for controlling the The study was conducted from April to May 2017. All Laiza district areas within 2.5 km from the border with China were covered, including urban and suburban regions of Laiza City, six surrounding villages , five distant villages , four internally displaced persons (IDP) camps , one school and two army camps. The total population included was 23,169 individuals residing in brick concrete bungalows in Laiza City, wooden tile houses in villages, and temporary sheds made of iron sheets and asbestos tiles in IDP camps. The study areas were depicted in Fig. Plasmodium falciparum (Pf)/Pan-LDH rapid diagnostic test (RDT) was the product of Guangzhou Wondfo Biotechnology Co., Ltd., China (lot W05460602WC). CLIP-PCR 2.0 kits were purchased from Diacurate Technology Co., China (Daanrui@126.com), and multi-section CLIP-PCR (mCLIP-PCR) kits for Plasmodium genus and species identification were donated by Peking Union Medical College, Beijing. The related probe sequences are provided in Additional file 1: Table S1. An Omron non-contact infrared forehead thermometer (model MC-720) was the product of Kunshan Reying Photoelectric Co., Ltd. (China), a real-time fluorescence quantitative PCR (qPCR) instrument (model CG-5) was purchased from Shanghai Likang Biomedical Technology Holding Co., Ltd. (China), and a shaking incubator (VorTemp 56) was obtained from Labnet International (USA).2 on the first floor of the outpatient department in the Nabang township health clinic . Worktops covered with ceramic tiles were positioned along the three walls and in the middle of the room. A two-person single-side vertical laminar flow biosafety cabinet was set near the front window at the entrance, and a standard refrigerator was placed to the right of the biosafety cabinet. The layout of the PCR laboratory is shown in Fig. A PCR laboratory was established in a single room of 20 mSampling was performed by seven trained healthcare workers, who notified the target community one day before the procedure. On the sampling day (day 0), a short community meeting was held to read out the informed consent form and deliver information on malaria and the associated health issues. Individuals were invited to participate voluntarily and signed informed consent. After registration of the blood test form, the body temperature of each participant was measured with a non-contact infrared forehead thermometer and two blood spots were collected with a trifold Whatman 903 Specimen Collection card. The blood spots on the open trifold card were dried briefly in a ventilated room by hanging the card on a rope for 30–120 min. The cards were then folded and brought to the laboratory in a special storage box on the same day. If a participant had body temperature ≥ 37.8 °C or reported fever history within the past week, a rapid diagnostic test (RDT) was performed at the time of blood sample collection and a blood smear was prepared in case of a positive result.Plasmodium spp. using CLIP-PCR with a matrix pooling strategy [Plasmodium. The solution was incubated with shaking (800 rpm) for 120 min at 56 °C to capture the target onto the plate. After plate-washing the bound detection probes on the target were ligated at 37 °C for 10 min to form a template for subsequent qPCR amplification in the same well with SYBR Green chemistry, using primers specific for the genus probes. For positive raw-pools and column-pools, their “intersection” samples were candidate positives and were further confirmed by assaying the individual samples without pooling, using CLIP-PCR for either Plasmodium spp. or Plasmodium genus and species (see below), while others were declared negative.Four technicians of the testing group placed the blood spot samples collected by the sampling group on the laboratory worktop for overnight drying. The next day (day 1), two dried blood-containing discs 3 millimeters (mm) in diameter were punched from one blood spot of each participant, and screened for strategy , 7. BrieP. falciparum 3D7 lysate was used as kit positive control and was placed randomly in the plate; several no-template controls were used as negative controls placed in wells next to the positive control. As an internal control to validate CLIP-PCR, blood smears of Day 0 RDT-positive participants were first confirmed by microscopy according to WHO recommendations [Throughout screening cultured ndations , 22, theP. falciparum was reported in the preceding two years were analysed for genus and species determination by multi-section CLIP-PCR (mCLIP-PCR) [Plasmodium genus-specific, P. falciparum species-specific and P. vivax species-specific sections of 18 S rRNA onto the plate. After plate-washing and a 50 µL probe-ligation reaction, the plate was heated for 1 min at 90 °C to release the ligated products, and 5 µL was transferred to a new PCR plate well for genus identification by qPCR performed in a total volume of 25 µL/well containing primers specific for the genus probes. Genus-positive samples were further assayed for species identification by transferring another 5 µL to a new well for qPCR, either with primers for the P. falciparum species-specific probes, or with primers for the P. vivax species-specific probes.The positives from areas where LIP-PCR) . BrieflyOn day 2, positive CLIP-PCR or mCLIP-PCR results were provided to a clinician who was responsible for the follow up of the participants. An additional RDT was performed on these positive participants to confirm some of the positives, and to see how many of these positives were below the detection limit of RDT. Regardless of the RDT results treatment was initiated to each positive participant according to WHO recommendations.The database was generated using Microsoft Excel 2010 and analyzed using descriptive statistics (SPSS Statistics version 20). The daily testing capacity was expressed as the number of CLIP-PCR-screened participants per day. The screening cost per person was determined as the actual purchase price of CLIP-PCR reagents divided by the number of screened participants. The CLIP-PCR-determined parasite prevalence rate (%) was calculated as the number of positive results divided by the total number of screened participants. In RDT confirmation of PCR results, the samples were regarded as microscopic when RDT results were positive and submicroscopic when RDT results were negative. The percentage of submicroscopic infections was calculated relative to the number of positive samples.P. vivax and 1 for P. falciparum by microscopy on Day 1.A total of 15,038 participants were sampled from April 26 to May 23, 2017, with a population coverage rate of 64.91% . There were 7260 men and 7778 women; the average age of the participants was 24.34 ± 17.66 years . On Day 0, a total of 118 individuals were tested by the RDTs. Among them 75 had fever (body temperature ≥ 37.8 ℃) on site and 43 had fever history within one week, and 9 of the 118 were found RDT-positive. Among them, 8 were tested positive for A total of 204 positive samples were identified among the 15,038 blood samples using CLIP-PCR kits for 35 plates. The PCR-based parasite prevalence rate was 1.36% , the testing capacity was 537 samples/day , and the screening cost was about US$ 0.92 /person . The 9 RDT-positive samples were confirmed by CLIP-PCR. The sensitivity and specificity of CLIP-PCR compared with microscopy were both 100%.P. vivax in the preceding two years and, therefore, individually-confirmed CLIP-PCR positive samples from these areas were considered P. vivax infections without further testing. The 53 CLIP-PCR positive samples from areas where P. falciparum was reported in the preceding two years were analysed for genus and species determination by multi-section CLIP-PCR (mCLIP-PCR). Among them, 53 were confirmed positive at the genus level by mCLIP-PCR with an agreement rate of 100% compared with CLIP-PCR. The species identification with mCLIP-PCR was 35 P. vivax, 14 P. falciparum, 2 P. vivax/P. falciparum mix and 2 negative. The 9 RDT-positive samples were identified at the species level by mCLIP-PCR with the same results as microscopy.Most of the surveyed areas in this study reported only P. falciparum or mixed species positive and 63 were P. vivax- or other non-P. falciparum positive . Among the 72 RDT positives, 9 were confirmed P. vivax, P. falciparum, and mixed P. falciparum + P. vivax, respectively. The proportion of P. vivax-infected participants was 92.2% and the ratio of P. falciparum to P. vivax infection was 0.09. The geographical distribution of malaria is summarized in Table The 204 patients screened positive with CLIP-PCR received treatment, among them, 188, 14, and 2 were treated as For decades since the invention of PCR, most molecular diagnostic innovations focused exclusively on amplification and detection to improve the sensitivity and specificity, but few focused on preanalytical process to reduce the complexity of molecular assays, which is the real impediment to their widespread applications. CLIP-PCR is conceptually similar to ELISA, with the capture probe and detection probe being analogous to the capture antibody and detection antibody. Subsequent PCR amplification is a signal amplification (rather than target amplification) analogous to the enzymatic cascade for signal detection in ELISA. Furthermore, the ligation step in CLIP-PCR affords two important benefits ELISA cannot have: (1) significantly better specificity and reduced background, as individual probes will not generate signal; and (2) ability to significantly avoid contamination, as it is only the ligated probes that are being detected. The reduced complexity of CLIP-PCR makes it possible to achieve high-throughput and sensitive molecular screening.Contamination, high cost associated with tedious procedures, and low throughput are some of the challenges in PCR-based detection. Therefore, the WHO requires that a laboratory performing PCR analyses with diagnostic purposes be divided into at least three physically separated compartments for reagent preparation, sample preparation, and amplification and product detection , 23. In Compared with “ordinary” PCR with pooling , 27, whi2 = 0.81) between Cq values of the two methods [The performance of CLIP-PCR, nested PCR, and quantitative reverse transcription-PCR (qRT-PCR) have been evaluated by Zhao et al. in testing of 1,005 individual samples collected from Laiza City and surrounding areas in 2015 . The aut methods .P. falciparum 3D7 lysates), as well as 9 RDT-positive samples identified on day 0 in the same population, was used. The RDT positives included 8 confirmed positive P. vivax and 1 confirmed positive P. falciparum by microscopy on day 1. Blind screening with CLIP-PCR indicated 100% positivity, and mCLIP-PCR identified the expected species.As both CLIP-PCR and mCLIP-PCR were originally validated with standard qPCR using dried blood spots , 9, and P. vivax outbreak in 2016 [P. falciparum/P. vivax ratio was 0.09, which are indicative of a very low transmission area according to the WHO categorization [P. vivax infection reported by Moreira et al. [As shown in Table rization . The proa et al.  and Chena et al. , indicatThis study has some limitations. Only 64.9% of the target population was covered by the screening. Although the sample collection team informed the target community one day in advance, some residents remained unwilling to volunteer, whereas others could not come in time, especially those who often work in the forest and are therefore at a high risk. This may have caused sampling bias. These results indicate that MSAT needs efficient logistics support. Furthermore, the PCR laboratory was established on the Chinese side of the border because of safety concerns, accommodation of researchers, and overall better conditions than in the Myanmar area under study. Although the laboratory was only 200 m away from some of the collection sites, it was not located in the field per se. Lastly, the rate of submicroscopic infection was calculated based on RDT results not on microscopy analysis, the actual number may be smaller.A strategy to include CLIP-PCR in MSAT for low transmission settings has the advantages of improved diagnostic sensitivity, high throughput, overall cost effectiveness, and timeliness of diagnosis and treatment. All these factors can be potential improvements to conducting MSAT in low transmission areas to support malaria elimination efforts in the GMS region.Additional file 1: Table S1. Probe sequences.


Escherichia coli (AIEC) has been implicated as a microbiological factor in the pathogenesis of inflammatory bowel disease (IBD). We evaluated the ability of six live biotherapeutic products (LBPs) to inhibit the interaction of an AIEC strain to three cell lines representing human gut epithelium. Co-inoculation of LBPs with AIEC showed a reduction in adhesion (up to 73%) and invasion of AIEC (up to 89%). Pre-inoculation of LBPs in HT-29-MTX and Caco-2 cells before challenging with AIEC further reduced the adhesion and invasion of the AIEC, with three LBPs showing significantly (p < 0.0001) higher efficiency in reducing the adhesion of AIEC. In co-inoculation experiments, the highest reduction in adhesion (73%) of AIEC was observed in HT-29-MTX cells, whereas the highest reduction in invasion (89%) was seen in HT-29-MTX and the co-culture of cells. Pre-inoculation of LBPs further reduced the invasion of AIEC with highest reduction (97%) observed in co-culture of cells. Our results indicated that whilst there were differences in the efficacy of LBPs, they all reduced interaction of AIEC with cell lines representing gut epithelium. Their efficiency was higher when they were pre-inoculated onto the cells, suggesting their potential as candidates for alleviating pathogenesis of AIEC in patients with IBD.Adherent-invasive  Escherichia coli has been implicated as a microbiological factor in disease pathogenesis [E. coli among patients with UC, CD and CRC [E. coli strains were designated adherent-invasive E. coli (AIEC). AIEC also carry several virulence genes not typically found among commensal E. coli strains [Inflammatory bowel disease (IBD) is a complex chronic idiopathic condition characterized by chronic bowel inflammation . It consogenesis . Several and CRC . Phenoty and CRC . These b strains .Lactobacillus strains and the gut epithelium has been shown using T84 cells serving as an epithelial barrier model, where Lactobacillus strains modulated the transcription of E-cadherin and β-catenin involved in maintaining adherence junction complexes [Lactic acid bacteria and, in particular, lactobacilli can provide significant beneficial roles in supporting the balance of a healthy gut microbiota by maintaining gut barrier integrity, supporting healthy digestive and immune function and suppressing the growth and adhesion of pathobionts ,14. Intromplexes . Furtheromplexes ,20. Theyomplexes .Candida spp. and Clostridioides (Clostridium) difficile infections with subsequent disease flares, it is critical to reduce the prescription of broad-spectrum antibiotics when possible [It is generally accepted that intestinal microbiota and/or their metabolic products, are likely an important factor involved in the pathogenesis of IBD. It is also accepted that treatment with antimicrobials to alleviate symptoms can also be associated with side-effects, such as bacterial overgrowth . With thpossible ,24,25. Fpossible . This coLactobacillus strains believed to be novel candidates as LBPs to colonize gut epithelium and to competitively exclude AIEC strains. We hypothesized that such competition will consequently inhibit or reduce the rate of AIEC colonization of, and invasion into, the intestinal epithelial cells and, therefore, they can be used as either a prophylactic or treatment option to prevent or alleviate symptoms associated with IBD. However, most studies assessing the interaction of LBP candidates utilize only one epithelial cell line which may not completely mimic the characteristics of the gut epithelium. Here we validated the efficacy of our LBP candidates to inhibit or reduce interaction of the AIEC strain F44A-1 with two cell lines i.e., Caco-2 and HT-29-MTX cells. These cell lines are derived from human colon adenocarcinoma and have been successfully used in many in vitro studies. Caco-2 cells can be differentiated in the culture medium to form a polarized cell monolayer with tight junctions and microvilli to resemble important characteristics of human intestinal mature enterocytes. The main drawback of this cell line is that it does not produce a mucus layer. The other cell line, HT-29, with methotrexate (MTX) adaptation i.e., HT-29-MTX cell line, differentiates in culture media to secret mucin [In view of the above, this study was undertaken to explore the ability of selected et mucin ,27,28. Met mucin . TherefoE. coli 46-4 was sourced from animals subjected to starvation and hemorrhage [Lactobacillus strains SVT 01D1, SVT 04P1, SVT 05P2, SVT 06B1, SVT 07R1 and SVT 08Z1. The AIEC strain was maintained at −80 °C in Luria-Bertani (LB) broth  with 20% glycerol. It was streaked on MacConkey agar first to check for its purity before growing on nutrient agar as a working culture and regrown in LB broth in a reciprocal shaker (140 strokes min−1) at 37 °C for 24 h before each adhesion or invasion assay. All LBP strains were maintained at −80 °C in de Man, Rogosa, Sharpe (MRS) broth  with 20% glycerol. They were grown on MRS agar as a working culture and regrown in MRS broth for 48 h at 37 °C before each assay.The AIEC strain F44A-1 was previously isolated from a patient with colorectal cancer and carried all phenotypic and genotypic characteristics consistent with AIEC strains including the presence of AIEC-associated virulence genes, their diffuse adhesion pattern to Caco-2 cells as well as survival and replication in macrophages . The lowmorrhage  and serv® HTB-37) alone, and HT-29-MTX-E12 cells (ECACC 12040401), originally differentiated from the HT-29 (ATCC® HTB-38) cell into mucin producing mature goblet cells using methotrexate (MTX) [v/v) fetal bovine serum (FBS)  for Caco-2 cells and 15% (v/v) FBS for HT29-MTX cells and 1% (v/v) penicillin-streptomycin . Co-culture of Caco-2 and HT-29-MTX cell lines was prepared by seeding Caco-2 and HT-29-MTX cells at a ratio of 9:1 and grown to confluence. The monolayer formed in this way could secrete a sufficient amount of mucin whilst expressing tight junctions and microvilli brush borders similar to that of the GI epithelium [Two human colon adenocarcinoma cells, Caco-2 cells (ATCCte (MTX) , as wellithelium .2. Culture media was changed every 48 h. At confluence, cells were sub-cultured into the eight-well chamber slides (Nunc Lab-Tek II) for adhesion assays and into sterile 96-well flat bottom plates for invasion assays.All cell cultures were maintained at 37 °C in an atmosphere of 5% CO−1) and LBP strains were cultured in MRS broth  for 18 h at 37 °C. Bacterial suspensions were centrifuged at 3500 rpm for 12 min and the supernatant was discarded. The pellets were resuspended in phosphate-buffered saline (PBS) (pH 7.4), and 100 μL of the original suspension  was inoculated into the appropriate chambers to give a multiplicity of infection (MOI) of 100 after counting the number of cells in three wells and adjusting the concentration of bacterial suspension.The adhesion of AIEC alone, LBPs strains alone, and AIEC in the presence of the LBP strains was tested on both cell lines and their co-culture using methods described previously , with mo9 CFU.mL−1 prior to incubation at 37 °C for 90 min. To assess the ability of LBP strains to prophylactically inhibit adhesion of AIEC, 100 µL of each LBP suspension (109 CFU mL−1) were pre-inoculated for 60 min onto the wells before infecting cells with the same concentration of AIEC. Cells were then incubated at 37 °C for a further 90 min and non-adherent bacteria were removed by washing the wells three times with PBS (pH 7.4). Cells were then fixed with 95% ethanol (v/v) for 5 min and stained using Gram staining to differentiate between Gram-positive LBPs (Lactobacillus strains) and Gram-negative AIEC and their adhesion was observed under a light microscope. The extent of colonization of the gut epithelium by both AIEC and LBP strains was determined by randomly selecting 100 cells and counting how many showed bacterial adhesion, while the ability of the strains to interact with each cell was determined by counting the bacterial number on 25 randomly selected cells showing adhesion.For the competitive adhesion, LBP strains and the AIEC were co-inoculated at an identical concentration, i.e., 10−1)  for 60 min to kill any extracellular bacteria and the contents were removed and washed three times with PBS and lysed with 0.1% (v/v) Triton X-100 (Sigma-Aldrich) to release the invading AIEC. The lysate was then serially diluted and 100 μL volumes were plated onto MacConkey Agar No. 3  plates and incubated for 24 h at 37 °C before colonies were counted. Mean ± SEM number of bacteria were calculated with due corrections for the dilution factors. E. coli strain 46-4 was used as a negative control for both assays at the same concentration of AIEC.For the invasion assay, a similar procedure was used as before  with somp < 0.05.All adhesion and invasion experiments were carried out in triplicates. GraphPad Prism statistical software (Version 8.0.0) was used for statistical analysis. Two-way ANOVA followed by Tukey’s multiple comparisons test were used to determine the differences in the mean level of adhesion and invasion among strains across all test groups, and the unpaired t test was used for comparing the difference between co-inoculation and pre-inoculation of each LBP. Correlation between adhesion and invasion capabilities of strains were evaluated using Pearson’s correlation coefficient. Differences were considered statistically significant if p < 0.0001) reduction in AIEC colonization of monoculture and co-culture cells, with a generally higher reduction in pre-inoculation experiments (p = 0.015), SVT 07R1 (p = 0.027) and SVT 08Z1 (p < 0.0001) significantly reduced the colonization of AIEC in the Caco-2 cells following co-inoculation (p < 0.0001) (p = 0.01 for both) between the co-inoculation and pre-inoculation experiments  . Pre-inoeriments .p < 0.0001) in the presence of most LBPs in both co-inoculation and pre-inoculation experiments (p < 0.0001) and Caco-2 cells (6.9 ± 0.5) (p = 0.0066) than to the co-culture model (4.2 ± 0.2)  .p = 0.02) and SVT 07R1  both significantly impaired invasion. All the LBPs showed a reduction following pre-inoculation when compared with co-inoculation results, however this again was cell-line dependent  between the number of AIEC adhering per cell and the number invading in the co-inoculation experiment but not in pre-inoculation (0.07) . This, hThe etiology of IBD, whilst not completely understood, is multifactorial in nature involving genetic susceptibility, external environmental factors, defective immune responses and alterations in the intestinal microbial composition ,39,40. ALactobacillus candidates reduced the adherence of AIEC to two cell lines that represent the intestinal epithelium, as well as their co-culture. This is supported by previous studies where Lactobacillus strains were shown to inhibit cell association and adhesion of some pathogens such as enterovirulant E. coli, Enterococcus faecalis and Salmonella typhimurium to Caco-2 cells [The mechanism of action of the LBPs may include the ability to effectively adhere to the intestinal mucosa and inhibit/reduce the ability of AIEC strains to adhere and colonize the gut epithelium, providing clinically meaningful outcomes. In this study, we demonstrated that the selected LBP -2 cells ,46.The AIEC strain used in this study was shown to be a highly adherent and invasive strain in our previous study via the presence of virulence genes typically found among AIEC strains, as well as its ability to survive and replicate within macrophages . This AIL. casei resulted in a decrease in AIEC LF82 adhesion to intestinal cells [In testing the ability of LBPs against AIEC, we used two methods of assessment. In co-inoculation studies, we aimed to evaluate the ability of LBPs to competitively exclude the pathogen-a mechanism that may lead to treatment of the IBD. We also assessed the ability of the LBPs to prophylactically inhibit colonization of the pathogen by pre-incubating cell lines for a period of 60 min to allow for an initial colonization of the LBP before challenging them with the pathogen. Time dependent inhibition of pathogen adhesion is a key trait, especially if they are to be used as prophylactics. This has been shown previously where pre-inoculation with al cells . In the To be of clinical benefit to the host, LBPs are expected to modulate intestinal pathobiont abundance and activity and protect the GI tract from environmental pathogens. Production of mucin by goblet cells in the GI tract is important to replenish and maintain the mucus barrier; however, this function can be disrupted by bacteria and their toxins. This can lead to pathological conditions like chronic inflammatory diseases . In co-iIn conclusion, we showed that the LBP candidates tested in this study significantly reduce the adhesion and invasion of the AIEC F44-1 strain in cell lines used although this efficacy varied among the strains. Pre-inoculation of the cell lines with the LBP candidates followed by the challenge with this pathogen was in most cases associated with a further reduction in adhesion and invasion of AIEC, although in some cases, this was cell line dependent. Further tests are needed to evaluate the potential of these strains before they can be introduced as an option for treatment of IBD.


Hereditary angioedema (HAE) is a rare, life-threatening disease. The knowledge about the molecular pathogenesis of HAE has derived mainly from investigating blood samples. However, limited data are available on the role of the molecular mechanisms in the affected tissues during HAE attack.The aim of our study was to explore the histological changes occurring in HAE attacks.Post mortem macro-, microscopic and immunohistological assessment of upper airway tissues of a patient with HAE due to C1 inhibitor deficiency (C1-INH-HAE) type 2 who died from laryngeal HAE attack was compared with a non-HAE patient who died from other condition without any signs of angioedema.Compared to the control patient, we demonstrated stronger T cell/monocyte infiltration and a more intense C1-INH staining in the C1-INH-HAE patient. The expression of both bradykinin receptors (B1/B2) was observed with a slightly lower level in the C1-INH-HAE patient than in the control patient. PAR1 expression was strongly reduced in the C1-INH-HAE patient suggesting overactivation of this hyperpermeability inducing receptor.Our unique case and novel results correspond to the knowledge about C1-INH and BDKRs observed in plasma; however, it revealed new information about the pathomechanism of HAE attack focusing on the potential involvement of PAR1 in edema formation. This observation, if it is verified by subcutaneous biopsy studies, may designate a new therapeutic target in HAE. SERPING1 gene and belongs to the bradykinin mediated angioedemas  concentrate) for relieving HAE attacks. She has undergone regular follow up at our Center. She has been experiencing limb edema once or twice a year, but airway attacks have never occurred. At the last follow-up visit 0.42 g/L C1-INH antigenic level was measured , 32% C1-INH functional activity , 0.02 g/L C4  and 119 mg/L C1q . One week after the last follow-up visit, progressive facial edema developed and preceded the onset of laryngeal edema. The patient dismissed these symptoms as non-serious and did not administer the medicinal product supplied for emergency use (pdC1-INH concentrate was available at her home), and died at home. Autopsy confirmed asphyxiation by laryngeal edema as the cause of death.The death of this 37-year-old female patient resulted from end-stage renal failure due to bilateral kidney atrophy caused by glomerular renal disease, complicated hypertension, acute heart failure, and circulatory insufficiency. Neither her family history nor clinical diagnosis showed any sign of C1-INH deficiency. Medical history was negative regarding angioedematous episodes and autopsy report did not describe any angioedematous tissues/organs.The study protocol was approved by the institutional review board of Semmelweis University of Budapest (Reg. No. 1067-5/2018/EUIG) in accordance with the Declaration of Helsinki.A complete autopsy was performed within 8 h after death with particular attention to the respiratory tract. Macro photos were captured by a digital camera (Fujifilm FinePix S5800). The larynx, the epiglottis, and the upper part of the trachea was removed and further processed as follows.A sagittal section of the whole larynx was placed in a macro-cassette. After gross sectioning of areas of interest for histological analysis and immunohistochemistry , tissue processing was performed, and after the appropriate duration of fixation in 10% neutral buffered formalin, samples were embedded in paraffin.Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) were performed on 4 μm-thick sections of the formalin-fixed, paraffin-embedded tissue blocks. Sections were deparaffinized by xylene and rehydrated in a graded series of ethanol and water. H&E staining was made using an automated stainer  and glass coverslipper . IHC was performed after endogenous peroxidase blocking in 0.01% periodic acid followed by 0.01% sodium borohydride and 3% hydrogen peroxide in methanol. Antigen retrieval was carried out for 30 min in a pressure cooker . The slides were incubated with the primary antibodies  and scored on a semi-quantitative scale of 0 to 3 based on the percentage of the positive cells and the staining intensity.In the HAE patient’s laryngeal sample, mucosal lining of the bronchi and trachea was non-hyperemic; it was covered by a small amount of airway secretion. The vocal cords were closed. A ball of froth was visible in the laryngeal introitus. The exploration of the larynx revealed intact vocal cords. The mucosa of the vestibular folds, the anterior and posterior aspects of the epiglottis, as well as the hypopharynx were edematous and swollen. The lumen of the esophagus was of normal size, its wall was of normal thickness, and its mucosa was intact Fig. .Fig. 1PhIn the control sample, the larynx and the trachea were of normal capacity, their lumen was patent. The esophageal lumen was of normal size; the mucosa was pale.To descript the exact location of the edema in the HAE patient, which directly caused suffocation, we applied routine histology staining. In HAE patient, hematoxylin–eosin staining revealed an extended edema at the epiglottis and the false vocal cord, whereas it was less pronounced at the areas of the true vocal cord and the trachea. Epithelial surface was substantially eroded at the epiglottic area. No similar edematous area or epithelial injury could be observed in the sample of the control patient Fig. . The micIn our previous studies, we found elevated white blood cell count in HAE patients compared to healthy controls . Based oNext, we asked what the histological pattern of C1-INH was in HAE patient, and whether this pattern or the intensity of C1-INH staining was different from that of the control. Since the HAE patient had a C1-INH-HAE type 2 disease with normal C1-INH concentration but with decreased C1-INH function, we expected moderate difference in C1-INH concentration compared to the control patient. Indeed, a weaker overall C1-INH signal was detected in the control as opposed to the HAE patient Fig. A, B.Fig.The pattern of C1-INH staining was similar in the HAE- and control patient, i.e. interstitial space, vessel lumen, epithelial cells (including glandular tissues), endothelial cells (ECs), muscle cells and leukocytes were intensively stained, whereas the examined cartilage tissues were negative at all four locations  (data not shown).Bradykinin receptors have great importance in HAE since they mediate the edematogenic response of bradykinin towards endothelial cells. Both receptors  were expressed in epithelial cells, leukocytes and ECs Fig. C–F. AlthWe recently showed that several activated plasma serine proteases, which can be inhibited by C1-INH, induce elevated EC permeability in vitro, predominantly via the PAR1 signaling pathway. We observed significantly lower expression of PAR1 in HAE patient than in the control sample. Interestingly, this difference was not restricted to the ECs and epithelial cells, but muscle cells were also included Fig. A–I. We dThis is the first immunohistochemical comparison of the laryngeal tissue of a patient with C1-INH-HAE who died from suffocation caused by upper airway angioedema with a control patient who died from other condition without any signs of angioedema. We found strong C1-INH staining in both patients—i.e., in the C1-INH-HAE and control patient-, besides a slightly decreased BDKRB1 and BDKRB2 expression and more intensive T cell/monocyte infiltration in the C1-INH-HAE patient. Interestingly, PAR1 expression was significantly lower in the C1-INH-HAE patient, which may reflect to receptor consumption due to PAR1 activation.post mortem specimen from the larynx of a C1-INH-HAE patient who succumbs to suffocation from laryngeal edema. Specifically, UAE is rare in C1-INH-HAE—occurring in approximately 0.9% of all AE attacks—and the proportion of patients who have ever experienced UAE during their lifetime is 50%  may further worsen the patient’s condition—especially in the case of submucosal edema formation , our obsrk et al. and our rk et al.. ConsequConsidering the macroscopic picture of laryngeal edema, we found that submucosal edema localized mainly at the epiglottic and false vocal fold areas, and the true vocal folds were less involved. These findings are similar to the previous observations . HematoxWe demonstrated moderate leukocyte infiltration in the C1-INH-HAE patient’s laryngeal tissue. We previously described that the level of several homing chemokines was elevated during HAE attacks , which mOur patient with type 2 C1-INH-HAE had an elevated C1-INH plasma concentration. In line with this, we demonstrated a slightly more intense C1-INH staining in the C1-INH-HAE patient than in the control patient. The staining pattern showed that endothelial cells, epithelial cells, muscle cells, leukocytes and interstitial space all contain a significant amount of C1-INH, which suggests that—besides the liver as the primary source of C1-INH production—the other tissues/cells are also able to produce and/or store C1-INH. These data are in line with the previous findings –18, whichttps://www.proteinatlas.org/ENSG00000100739-BDKRB1, https://www.proteinatlas.org/ENSG00000168398-BDKRB2). In our cases, both bradykinin receptors were expressed in the C1-INH-HAE patient and in the control patient, too. The expression of both bradykinin receptors was slightly lower in C1-INH-HAE patient than in the control patient. This suggests that both bradykinin receptors were activated during the HAE attack of our patient.We found an interesting and surprising staining pattern regarding the bradykinin receptors. The literature is controversial in this field: BDKRB2 was considered as the constitutive and BDKRB1 as the inducible bradykinin receptor , althougOur most important and novel observation was that the PAR1 expression was strongly reduced in the C1-INH-HAE patient compared to the control patient. In our previous in vitro study, we showed that PAR1 activation by thrombin, MASP-1  and otheFinally, if our observations, derived from the sample of a type 2 C1-INH-HAE patient, are validated, they may well be applicable for the pathomechanism of type 1 C1-INH-HAE and probably (with some restrictions) for that of other BK mediated angioedemas.In conclusion, studying the molecular and cellular mechanisms in human angioedematous tissues is a necessary future target besides blood plasma analysis. On the one hand, our unique case and novel results confirmed those observed in plasma , on the other hand, it suggested new information about the pathomechanism HAE attack, which seems to be much more complex than we had thought before.


Mycoplasma hominis, which is difficult to culture and identify by ordinary methods, is one of the smallest pathogens in the human genitourinary tract causing urogenital infections. A CRISPR-Cas12a-based detection system might provide a novel application for M. hominis nucleic acid detection in molecular diagnostics. A plasmid containing the glyceraldehyde-3-phosphate dehydrogenase gene of M. hominis (ATCC_27545) as the positive control was constructed by homologous recombination. The active Cas12a protein was purified by affinity chromatography. The primers for recombinase polymerase amplification (RPA), the CRISPR RNA (crRNA), and the ratio of Cas12a to crRNA were further optimized. Finally, the sensitivity, specificity, and clinical effectiveness of the Cas12a detection system were confirmed. We successfully constructed and optimized a novel nucleic acid detection system for M. hominis based on RPA-CRISPR-Cas12a, and the whole process takes only 1 h. The limit of detection for the gap gene of M. hominis was 3 copies/μl and no cross-reactivity with other urogenital pathogens appeared. In the evaluation of 111 clinical samples, the sensitivity and specificity were both 1.000 and the area under the curve of the receiver operating characteristic was 1.000 (p < 0.001), indicating that the RPA-Cas12a-fluorescent assay was fully comparable to the traditional culture method. Finally, the RPA-Cas12a detection system can also be combined with lateral flow strips (LFS) to achieve visual detection. We successfully developed a low-cost and rapid detection method of M. hominis based on RPA-Cas12a technology. This method realized by fluorescence value readout and visual detection by LFS could be applied in population screening and resource-limited conditions. Mycoplasma hominis nucleic acid detection based on the CRISPR-Cas12a system. Clinical samples from cervical or urethral swabs were collected and incubated with lysis buffer to release nucleic acid (10 min). Extracted DNA (1 μl) is subjected to the RPA reaction with specific primers at 37°C. After 20 min, RPA product was subjected to the CRISPR-Cas12a reaction for cleavage. The collateral nuclease activity of Cas12a (250 nM) proteins were activated upon specific binding to crRNA  and the DNA product; thus, Cas12a cut the quenched fluorescent ssDNA reporter (125 nM) (30 min). The generated fluorescence signal would be measured by a fluorescence plate reader or visualized by lateral flow strips.Schematic of  Mycoplasma hominis, an obligatory parasite in human genitourinary tract, belongs to the class Mollicutes -associated nuclease system has provided potential applications for rapid and sensitive molecular diagnosis . The CRIBy specifically and accurately cutting dsDNA matched to its corresponding specific crRNA with a thymine nucleotide-rich protospacer-adjacent motif (PAM) sequence, Cas12a attains the capability to further cut ssDNA probes labeled with fluorescent tag or biotin . ReadoutM. hominis. Here, we provided the application of CRISPR-Cas12a detection in simple, rapid, sensitive, and reliable M. hominis diagnosis. Different RPA primers and crRNAs were tested for their efficiency, and the optimized RPA-Cas12a workflow was then evaluated for sensitivity and specificity. Visualized detection was also assessed for its compatibility with the assay. Finally, the performance of the RPA-Cas12a system was compared with the culturing method in the detection of M. hominis in clinical samples. The reaction process of Cas12a-based M. hominis DNA detection system is shown in the Graphical abstract.Cas12a is rarely used in the detection of urogenital pathogens, and there is no relevant report in the diagnosis of 1, and in vitro transcribed (IVT) templates for crRNA were ordered from Genewiz . Commercial Cas9, Cas12a, Cas13a, and FAM-TTTTT-Quencher used in fluorescent reporter assay and FITC-TTTTTT-Biotin probe used in gold nanoparticle-labeled lateral flow strips were obtained from Biolifesci . The detailed sequences are listed in M. hominis genomic DNA was extracted using lysis buffer for Microorganism to Direct PCR . The TwistAmp® Basic kit for RPA test was purchased from TwistDx . T7 RNA polymerase, the NTP mix, and the RNA purification kit were purchased from New England Biolabs . RNase-free water and Recombinant DNase I (RNase-free) were purchased from Takara. RNase-free water was used in all experiments. Streptavidin was purchased from Solarbio , while Goat anti-Rabbit IgG and gold-nanoparticles–Rabbit anti-FITC were purchased from Bioss . The fluorescence quantifications were measured with a Wallac 1420 plate reader . Amplification was confirmed by 3% agarose gel electrophoresis and the RPA products were visualized using a Gel Doc system . The relevant reagents are presented in Primers for RPA were designed using NCBI Primer-BLASTSpecimens obtained for testing included cervical or vaginal swabs for women and urethral swabs for men. A total of 178 samples were collected from sexually transmitted disease (STD) clinics at Shantou Second People’s Hospital in China from June 2020 to November 2020, including 29 samples from men and 149 samples from women. We collected two swabs from the same part of a patient, one for culture and one for nuclear acid detection with the RPA-CRISPR-Cas12a assay. Samples were processed within 2 h after collection. The study was approved by the Institutional Medical Ethics Review Board of Shantou Second People’s Hospital.g for 1 min. Bacterial cells were harvested by centrifugation. After removing the supernatant, 50 μl of lysis buffer for Microorganism to Direct PCR was added to the precipitates and heated for 10 min at 85°C according to the manufacturer’s instructions. The supernatant extracts were directly used for the RPA reactions of the M. hominis. A 280-bp fragment of the glyceraldehyde-3-phosphate dehydrogenase (gap) gene of M. hominis  and centrifuged at 14,000 ×  hominis  was ampl hominis  accordinIn order to get a satisfactory performance of the RPA reaction system, several parameters were supposed to be optimized, such as the concentration of the primers, reaction temperature, and incubation time. The concentration of primers was first optimized. We added 0.1, 0.25, 0.5, and 1 μM primers to the RPA reaction system, respectively  was obtained from the 6His-MBP-TEV-huAsCpf1 (AsCas12a) expression vector of Sail Health Biology. The fusion protein contained an N-terminal His6-MBP tag followed by a tobacco etch virus (rTEV) protease cleavage site. The AsCas12a protein was expressed in E. coli Rosetta2 (DE3) that were induced with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside  at OD600 = 0.6 for 16 h at 21°C in LB medium, supplemented with 34 μg/ml ampicillin. Cells were collected and lysed by sonication (1 s on and 2 s off) in buffer containing 20 mM Tris–HCl, pH 7.5, 1 M NaCl, 20 mM imidazole, and 10% glycerol. After centrifugation, the supernatant was incubated with nickel-nitrilotriacetic (Ni-NTA) resin . The bound protein was eluted with buffer containing 20 mM Tris–HCl, pH 7.5, 150 mM NaCl . The eluted AsCas12a protein was digested with rTEV protease  at 16°C overnight to remove the His6-MBP tag. We again combined the mixture with Ni-NTA. The miscellaneous proteins with His-tag (including Cas12a with MBP-tag and other miscellaneous proteins) would be combined with Ni NTA while the pure Cas12a was not combined with Ni NTA. After elution, the rest was the purified Cas12a. Furthermore, the purified Cas12a was concentrated to a concentration of 3 mg/ml on Amicon Ultra-4 Centrifugal Filter Unit (Merck-Millipore), with buffer containing 20 mM Tris–HCl, pH 7.5, 1 M NaCl, and 10% glycerol as eluent.The Cas12a nuclease derived from 2 and they were generated from a DNA template, which is the reverse complement of target crRNA sequences via in vitro transcription  in a 20-μl reaction volume (Genomic DNA extract (1 μl) was used as input for RPA reaction . The complete detection system of RPA-Cas12a-Fluo assays was performed with 250 nM purified AsCas12a, 62.5 nM crRNA3, 1 μl target DNA (the amplified RPA products), and 125 nM quenched fluorescent ssDNA reporter mixed in Cas12a reaction buffer . The clinical specimens were injected into the liquid medium and placed in the candle jar. After the culturing at 37°C for 24–48 h, the color of the culture tube was observed every day. The medium changing from yellow to pink or red suggests the growth of M. hominis.The culture of M. hominis negative clinical samples (13 from men and 54 from women) were tested based on the RPA-Cas12a-Fluo assay, and the average value and standard deviation were calculated. The fluorescence threshold of the RPA-Cas12a-Fluo assay was set as the average value plus three times the standard deviation. The result higher than the fluorescence threshold was determined to be positive. Subsequently, 111 clinical specimens (16 from men and 95 from women) were tested based on the RPA-Cas12a-Fluo assay and compared with the results of the liquid medium culture as a gold standard. The results of these two methods were analyzed for correlation and consistency.Sixty-seven 2, pH 7.3) in a 20-μl reaction volume. To analyze the results of Cas12a detection with LFS, 20 μl of the above mixture was added to 60 μl of PBS following loading onto the sample pad in LFS for 2 min. To determine the intensity of T lines, we converted the strip image to 8-bit grayscale and measured the gray values of the lines using ImageJ, and calculated the indicated relative T line intensity of clinical samples by subtracting them by the gray value of the negative control.Combining the RPA-CRISPR-Cas12a process with lateral flow strips, we further developed a RPA-CRISPR-Cas12a lateral flow strip assay (RPA-Cas12a-LFS). Similar to that reported in another study (15), the kind of LFS  is different from other conventional test strips. If the sample is positive, the C line and T line will both appear, while the C line will not appear with only the T line present when the sample is strongly positive. The reporter of ssDNA probe was labeled with fluorescein isothiocyanate (FITC) at the 5′ and biotin at 3′, respectively. The RPA-Cas12a-LFS assay was performed with 250 nM purified AsCas12a, 62.5 nM crRNA, 1 μl of target DNA (the amplified RPA products), and 125 nM ssDNA reporter labeled with FITC and Biotin mixed in Cas12a reaction buffer , normal urinogenital bacteria (including Staphylococcus aureus (ATCC 10832), Staphylococcus saprophyticus (ATCC 15305), Klebsiella pneumoniae (ATCC 10031), Escherichia coli (ATCC 25922), and other urinogenital tract pathogens  were detected to determine the specificity of the RPA-Cas12a Fluo assay and RPA-Cas12a-LFS assay, which were performed as described in Section “RPA-CRISPR-Cas12a-Based Fluorescent (RPA-Cas12a-Fluo) Assay” and Section “Lateral Flow Detection Reactions”, respectively.Since there are many bacteria present in the urinogenital tract, the swabs are prone to be contaminated with other bacteria during sample collection. To avoid interference by other normal urinogenital bacteria or urinogenital tract pathogens, we selected four normal urinogenital bacteria and six common urinogenital tract pathogens as the specificity evaluation. Eleven pathogens including target pathogen Statistical analyses were performed with GraphPad Prism 7. Each experiment was repeated three times for each sample. The initial fluorescence from all samples and the background condition (no templates) fluorescence were subtracted to generate background subtracted fluorescence. The results are presented as mean ± SD unless otherwise indicated. Regarding the liquid medium culture assay as a gold standard, the sensitivity of the developed assay was calculated as follows: sensitivity = true positive/, while the specificity was calculated as follows: specificity = true negative/.M. hominis-specific sequence , we added it to RPA amplification at 37°C for 20 min. The results of nucleic acid electrophoresis revealed that our RPA primer 2 were with remarkable specificity, which is conducive to the Cas12a detection system  containing Cas12a protein-coding sequence for prokaryotic expression and purified it properly with Ni-NTA resin. After elution, Cas12a with MBP-tag (approximately 196 kDa) was collected . Subsequigestion . The resigestion . After 4igestion  or inact7 copies per reaction. However, the combination of CRISPR-Cas12a and RPA, called the RPA-Cas12a-Fluo assay, achieved a notable limit of detection (LOD) of three copies of M. hominis DNA. Next, to evaluate the effect of primer concentrations in RPA, we added different concentrations of primers in RPA reaction  with the RPA-Cas12a-Fluo assay. As shown in M. hominis detection. Additionally, the ratio of Cas12a to crRNA was also optimized. AsCas12a:crRNA3 was set with varying proportions in the RPA-Cas12a-Fluo assay, and the fluorescence signals were collected. The subtracted fluorescence showed that a 4:1 ratio of Cas12a:crRNA3 (250 nM purified AsCas12a:62.5 nM crRNA3) ensured Cas12a-mediated cleavage with less amount of reactants consumed . The sensitivity and specificity were both 1.000  and the area under the curve (AUC) was 1.000 (To detect be 2,915 . Samplesnegative . Of the positive . Comparias 1.000 . Taken tM. hominis nucleic acid detection. The entire detection process can be completed within 1 h, including 10 min for releasing sample genomic DNA, 20 min for RPA replication, and 30 min for the CRISPR-Cas12a reaction and readout. According to the rationale of LFS, a red T line indicates a positive sample, while the absence of a red T line indicates a negative sample (M. hominis and other urogenital bacteria including C. albicans, E. coli, K. pneumoniae, S. aureus, S. flexneri, S. saprophyticus, U. urealyticum, C. trachomatis, N. gonorrhoeae, and M. genitalium. The T line only appeared under the pathogen of M. hominis, while this line could not be detected in samples of other pathogens analyzed (More importantly, in order to broaden the application of the Cas12a system, we performed this system with commercial LFS to realize the visual interpretation of results in e sample . We firsanalyzed .R2 was 0.8964 (Finally, to assess the efficacy of the RPA-Cas12a-LFS assay in clinical samples, 37 clinical specimens were randomly selected from 111 clinical specimens described above. These 37 clinical samples were both detected with RPA-Cas12a-Fluo and RPA-Cas12a-LFS assay. Strikingly, the results of the RPA-Cas12a-LFS assay were 100% consistent with the RPA-Cas12a-Fluo assay . We furts 0.8964 . Furthers 0.8964 .Enterocytozoon hepatopenaei. Compared with other Cas proteins that recognize and cleave RNA sequences such as Cas13a and Cas13b (M. hominis rapid nucleic acid detection, which only takes less than 1 h.The CRISPR system is an acquired immune defense system against nucleic acids in bacteria that includes many different Cas proteins with nuclease activity . Among td Cas13b , Cas12a d Cas13b . At the M. hominis and M. genitalium have high homology, especially the information of gap gene (M. hominis from M. genitalium. Therefore, it is of great significance for clinical detection.RPA-Cas12a assay developed in this study had no cross-reactivity with other urogenital pathogens and could consistently detect three copies of DNA per reaction. In addition, when we adopted RPA for specificity test, non-specific bands appeared in the results of nucleic acid electrophoresis. Since those bands were unexpected, we were worried that those bands will affect the subsequent detection at the beginning. Surprisingly, they did not affect our results in the subsequent experiment. Non-specific bands sometimes appear in high-density samples , whereasgap gene , making M. hominis. In order to ensure the amount of nucleic acid extracted and speed up the whole detection process, we used the direct lysis buffer with a fast nucleic acid extraction kit, which can quickly extract adequate nucleic acid within 10 min. Additionally, we optimized the components of the reaction system including the concentration of RPA primers, the selection of crRNA, and the ratio of Cas12a protein to crRNA. All of the efforts ensured that the whole reaction process can be completed within 1 h.Since the urinary tract swabs usually contain limited amount of bacteria, if it is extracted by boiling  simply, M. hominis diagnostics, we further applied commercial LFS to achieve the visual interpretation of results. The experimental results showed that the LFS detection preserved the high sensitivity of the RPA-Cas12a reaction with an LOD of three copies, and the specificity is also considerable. So far, we have established the RPA-Cas12a-LFS assay, which may be used as an auxiliary means of nucleic acid detection in clinical practice in the near future.Although the Cas12a detection system we built can be readily operated, considering its dependence on a fluorescence detective instrument that represents a barrier to the implementation of portable devices for M. hominis, including liquid medium culture, qRT-PCR, and ELISA. In M. hominis. Although these methods offer good sensitivity, they are limited by various constraints such as being time-consuming and lack of compatibility with point-of-need applications. For example, in spite of liquid medium culture being determined as the gold standard, it is still slow and laborious (M. hominis nucleic acid, it still needs several large laboratory instruments and, hence, more practical for laboratory settings than for field work (M. hominis (M. hominis. However, the CRISPR-Cas12a detection system takes less time and is more convenient for rapid screening. Also, once the sample is confirmed to be positive, the drug sensitivity test with liquid medium culture can be carried out, which is more cost-saving.Thus far, several detection platforms have been developed for aborious . As to qeld work . Althoug hominis , it is sU. urealyticum usually causes urogenital tract infection, and it is often accompanied by M. hominis infection (M. hominis and U. urealyticum infection, such as developing a multi-detective platform combined with other Cas proteins, which is more meaningful for clinical practice.Additionally, nfection . TherefoM. hominis RPA-Cas12a-LFS detection system with two output modes, which were fluorescence signal and strips. These two methods complement each other and greatly improve the detective accuracy, which are more persuasive and reliable for evaluating M. hominis infection and expected to be applied in clinical practice.Although the LFS with gold nanoparticles conjugated anti-FITC is very convenient to use, it is difficult to perfectly prepare gold nanoparticles with uniform size and single dispersion. Heterogeneous gold nanoparticles are difficult to uniformly coat the corresponding antibodies, which makes them prone to non-specific aggregation, and their own scattered light interference will also bring a high background signal . TherefoM. hominis infection with high sensitivity and specificity based on the RPA-CRISPR-Cas12a detection. The developed detection procedure requires minimal template with an LOD of 3 M. hominis copies, little amount of reagents, and a convenient reaction condition, and is time-saving, which meets the needs of clinical detection of M. hominis. The method did not exhibit false-positive results with other urogenital pathogens, and was robust to the presence of abundant background DNA from human. Also, we attempted to combine LFS with the RPA-CRISPR-Cas12a detection to get rid of the dependence on large instruments. Therefore, the RPA-Cas12a detection shows great promise for routine detection of M. hominis, and is highly accessible to population screening or resource-limited settings in molecular diagnostics.In this study, we demonstrated a novel detection method for the diagnosis of The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Medical Ethics committee of Shantou Second People’s Hospital. The patients/participants provided their written informed consent to participate in this study.JC, YH, and BX designed and performed the experiments. HD collected the clinical specimens. KG and KL performed the data analysis. WH wrote the manuscript. WH and LL conceived and designed the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Additionally, it could inhibit the expression of P-JNK in macrophages. However, the JNK activator anisomycin could increase the proportion of CD80+ macrophages. In addition, exosomes of the uterine aspirate fluid from endometriosis could promote the migration and invasion of endometrial stromal cells by acting on macrophages. The expression of miR-210-3p was increased in both exosomes and the eutopic endometrium in patients with endometriosis through miRNA sequencing, which could also reduce the proportion of CD80+ macrophages. In summary, we propose that exosomes from the uterine cavity in patients with endometriosis may affect the phenotype of macrophages by inhibiting the JNK signaling pathway, thus mediating the formation of an immunological microenvironment conducive to the development of endometriosis.Endometriosis is a chronic inflammatory disease with an uncertain pathogenesis. Peritoneal immune dysregulation plays an important role in the pathogenesis of endometriosis. Exosomes are messengers of intercellular communication. This study mainly investigated the role of exosomes from the uterine cavity in endometriosis. Exosomes of the uterine aspirate fluid were isolated and cocultured with macrophages for 48 h. Flow cytometry was used to detect macrophage polarization. A Human MAPK Phosphorylation Antibody Array and Western blot were used to detect the phosphorylation of the MAPK pathway. A microRNA sequencing analysis was used to detect differentially expressed miRNAs. Our research found that exosomes of the uterine aspirate fluid from endometriosis could reduce the proportion of CD80 Endometriosis (EMs) is a chronic inflammatory disease that can lead to chronic pelvic pain and infertility . It can Endometriosis has a contentious etiology which is often considered to be related to retrograde menstruation, coelomic metaplasia, and lymphatic and vascular metastasis . EndometExosomes are microscopic membrane vesicles with a diameter of 30–150 nm that are mainly composed of lipids, proteins, and nucleic acids . ExosomeExosomes are messengers of intercellular communication. Campoy et al. successfully isolated exosomes from the fluid fraction of uterine aspirates, which may reflect the physiologic status of the uterine cavity . The isoThis study was approved by the Ethics Committee of The Second Xiangya Hospital of Central South University (#2020-065). All the patients included in this study signed informed consent forms. The EMs group (n = 22) included patients who underwent laparoscopic surgery for ovarian endometriosis at the Second Xiangya Hospital of Central South University between January 2020 and August 2021. The average age of the EMs group was 29.32 years. All the patients with endometriosis were at stage III/IV according to the revised American Society for Reproductive Medicine classification system. The control group (n = 25) included patients who underwent surgical treatment for other benign gynecological diseases except for endometriosis, such as simple ovarian cyst, uterine fibroids, and tubal ligation. The average age of the control group patients was 28.44 years. All included patients had a regular menstrual cycle and had not taken any hormone drugs or been placed on an intrauterine device for at least 6 months before surgery. Patients with malignant tumors, autoimmune diseases, hyperthyroidism, severe liver and kidney insufficiency, infectious diseases, or psychiatric diseases were excluded.g for 20 min to remove endometrial tissues and cell debris. The endometrial tissues were stored at −80 °C for miRNA sequencing. The supernatant was filtered through a 0.22 μm filter  to obtain the uterine aspirate fluid. The uterine aspirate fluid was stored at −80 °C for exosome extraction.After the vulva, vagina, and cervix were routinely disinfected, a disposable uterine tissue suction catheter  was used to aspirate approximately 1 mL of uterine aspirates. Then, the uterine aspirates were diluted in equal proportions of phosphate-buffered saline (PBS). Next, the samples were centrifuged at 4 °C, 2500× g for 10 min with a 100 KDa Amicon Ultra Centrifugal Filter  to obtain exosomes.Exosomes were isolated using the exosome isolation kit . This kit is based on the principle of size exclusion chromatography (SEC) to separate exosomes. Yang et al. showed that SEC was more suitable for RNA sequencing in extracellular vesicles . We sepaA total of 10 μL of exosomes were dropped on a copper mesh. After incubation at room temperature for 10 min, the copper mesh was washed three times with ultrapure water. Then, 2% uranyl acetate was used to stain the exosomes for 1 min. The morphology of the exosomes was observed with a TEM  at 80 KV after the copper mesh was dried thoroughly.7 particles/mL to 1 × 109 particles/mL with PBS to detect the size and quality of the exosomes.The concentration of exosomes was measured with a 405 nm laser by ZetaView PMX 110 . Then, the concentration of the exosomes was diluted from 1 × 10In brief, the exosomes and collected cells were lysed with a RIPA lysis buffer  to extract the proteins. The total protein concentration was detected using the BCA Protein Assay kit . Then, the protein was separated using SDS-PAGE and was transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membrane was blocked with 5% skim milk powder at room temperature for 1 h. Next, the membrane was incubated with primary antibodies: anti-CD63 , anti-TSG101 , anti-HSP70 , anti-Calnexin , anti-JNK , anti-P-JNK , anti-CD36 , anti-SIRP-α , and anti-IL-4R  at 4 °C overnight. After washing with a TBST buffer three times, the membrane was incubated with the secondary antibody at room temperature for 90 min. Finally, the protein bands were visualized with enhanced chemiluminescence . The immunoreactive bands were quantified with Image J software .®Mini Kit  according to instructions. The quality and quantity of the RNA were analyzed using NanoDrop 2000 . miRNA sequencings were performed on an Illumina HiSeq2500 . The differential expression miRNAs were screened with a Fold change (FC) ≥ 1.50 and p < 0.05 as the criteria.The total RNA was extracted from exosomes and endometrial tissues using the miRNeasy−ΔΔCt method was used for statistics. The sequence of the primers and probe was as follows: hsa-miR-210-3p-RT: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGCC; hsa-miR-210-3p-F: CCTGTGCGTGTGACAGC; hsa-miR-210-3p-P: TTCGCACTGGATACGACTCAGCC; U6-RT: AACGCTTCACGAATTTGCGT; U6-S: CTCGCTTCGGCAGCACA; U6-A: AACGCTTCACGAATTTGCGT; U6 probe: AGAAGATTAGCATGGCCCCTGCGCA; qPCR-TYR: GTGCAGGGTCCGAGGT.The total RNA was reverse transcribed into cDNA with the PrimeScript™ RT reagent Kit  according to instructions. The real-time quantitative PCR was performed to detect miRNA levels with the TaqMan probe. The 2A human acute monocytic leukemia cell line (THP-1) was cultured in an RPMI-1640 medium  with 10% exosome-free fetal bovine serum . Then, THP-1 cells were treated with 100 ng/mL phorbol-12-myristate-13-acetate (PMA) for 48 h to differentiate them into naive macrophages (M0). The typical morphology of macrophages can be observed under a light microscope. Next, exosomes (100 μg/mL) were added and incubated with macrophages. In addition, to examine the effect of the exosomes on macrophages after JNK activation, JNK activator anisomycin  was coincubated at a concentration of 1 μg/mL for 48 h. Cells were collected for flow cytometry, Western blot, or MAPK Phosphorylation Antibody Arrays.First, the exosomes were stained with PKH67 . Then, the PKH67 green fluorescence-labeled exosomes were co-incubated with macrophages for 6 h. Next, the macrophages were washed with PBS twice and fixed with 4% paraformaldehyde for 30 min. DAPI was added to stain the cell nuclei for 10 min. The uptake of exosomes by macrophages was observed under a fluorescence microscope.Endometrial tissues of the control group were collected to isolate and culture endometrial stromal cells. First, the endometrial tissues were rinsed with PBS and cut into little pieces. Next, the tissues were digested with 2.5 mg/mL type I collagenase  and 0.1 mg/mL DNase I  for 30 min. The digested tissues were filtered through a 40 μm sieve. Then, the isolated endometrial stromal cells were resuspended in an RPMI-1640 medium with a 10% fetal bovine serum.4 cells suspended in a 200 μL serum-free RPMI-1640 medium were seeded in the upper chamber of the transwell chamber. The lower chambers were THP-1-derived macrophages, which were treated with exosomes (100 μg/mL) and cultured in an RPMI-1640 medium with a 10% exosome-free fetal bovine serum. After 48 h, the transwell chambers were removed and washed twice with PBS. Then, the migrated cells were fixed with methanol for 10 min and stained with 0.1% crystal violet for 30 min. The number of migrated cells were counted under the microscope. Cells in five fields per chamber were randomly selected for counting. In the transwell invasion assay, 1 × 105 endometrial stromal cells were seeded in the upper chamber of the transwell chamber, and the chamber bottom was precoated with Matrigel. The next experimental operations were consistent with the transwell migration assay.The endometrial stromal cells were starved with the serum-free RPMI-1640 medium for 24 h. In the transwell migration assay, 5 × 10THP-1-derived macrophages were cultured in an RPMI-1640 medium  with a 10% exosome-free fetal bovine serum . Then, miR-210-3p mimics, mimics negative control (NC), miR-210-3p inhibitor, and inhibitor negative control  were used to transfect macrophages with Lipofectamine 2000 . The transfection efficiencies were detected using RT-qPCR. After 48 h of transfection, the macrophages were collected for flow cytometry.g for 10 min. The total protein concentration of the supernatant was detected using the BCA Protein Assay kit . The supernatant was diluted with a cell lysis buffer to ensure that the total protein concentration of each cell lysis supernatant was 80 μg/mL. Then, the cell lysates were incubated in 2 mL of blocking buffer at room temperature for 30 min. Next, the blocking buffer was removed, and the protein samples were incubated overnight at 4 °C. After being washed three times with wash buffer I and wash buffer II, the membranes were incubated with a detection antibody cocktail for 120 min at room temperature. Then, the membranes were incubated with horseradish peroxidase-conjugated antirabbit IgG for 120 min at room temperature. The membranes were visualized with the chemiluminescence imaging system .A Human MAPK Phosphorylation Antibody Array  was used to detect the phosphorylation of the MAPK pathway in macrophages after exosome intervention. In brief, macrophages were washed with PBS twice after incubating the exosomes with macrophages for 48 h. Then, a cell lysis buffer was added to the lysate macrophages at 4 °C. After 30 min of lysis, the supernatant was collected by centrifugation at 14,000× The digested macrophages were washed twice with PBS. Then, the cells were centrifuged at 1500 rpm for 5 min to remove the supernatant, and then they were incubated with 5 μL of anti-CD80-FITC  at 4 °C for 30 min in the dark. Flow cytometry was performed using a CytoFLEX flow cytometer , and the cells were analyzed using CytExpert software .p < 0.05 is considered to be statistically significant. The student’s t-test was used for statistical analysis between two unpaired groups. A one-way analysis of variance (ANOVA) was used to compare differences between three or more groups.Statistical analyses were performed using GraphPad Prism 7.0 software . The data are shown as the mean and standard deviation, and The morphology of exosomes was observed using TEM. Exosomes showed a typical round and cup-shaped morphology A. MoreovPKH67 green-fluorescence-labeled exosomes and DAPI stained the macrophage nuclei A,B,D,E. + macrophages (M1 macrophages). The results revealed that exosomes of the uterine aspirate fluid in patients with endometriosis decreased the proportion of CD80+ macrophages compared with the control and PBS  ≥ 1.50 and etriosis A,B. RT-qetriosis C.p < 0.05 as criteria. A total of 1327 miRNAs were detected in the EMs group and the control group. Eighteen upregulated miRNAs  and four downregulated miRNAs  were found in the eutopic endometrium from patients with endometriosis compared with the control  ≥ 1.50 and  control A,B. miR- control C. The re+ macrophages was significantly decreased after transfection with miR-210-3p mimics compared with the PBS and miR-210-3p mimics-NC  ,36. RNA-Furthermore, our study showed that miR-210-3p mimics inhibit the M1 polarization of macrophages. This indicates that the exosomes from endometriosis that inhibit macrophage M1 polarization by inhibiting the JNK pathway may be closely related to exosomal miR-210-3p. Dai et al. discovered that the elevation of miR-210-3p can decrease the expression of BARD1, which contributes to the development of endometriosis . The exoExosomes in endometriosis not only affect the phenotype of macrophages but also affect the function of macrophages by affecting the expression of macrophage receptors. The expression of the macrophage receptor CD36 was downregulated after exosomes of the uterine aspirate fluid were taken from the endometriosis intervention. CD36 belongs to the Class B scavenger receptor family, which is closely related to the phagocytosis of macrophages . Chuang Exosomes were successfully isolated from uterine aspirate fluid. Exosomes from the uterine cavity were highly expressed miR-210-3p in patients with endometriosis. These exosomes could transmit signals from the uterine cavity to the abdominal cavity. Then, it could inhibit the M1 polarization of macrophages by inhibiting the activation of the JNK signaling pathway. The reduction in M1 macrophages in the peritoneal cavity could promote the migration and invasion of ectopic endometrial stromal cells . Exosome


IL-10 and the serum levels of IL-10 in patients with pituitary adenoma. Methods: Data from 106 patients with pituitary adenoma and 192 control patients were used for the study. DNA was isolated from peripheral blood using the salt precipitation method. The samples were genotyped in real-time using the polymerase chain reaction method. IL-10 serum levels were evaluated using an ELISA kit. The data obtained were systematized using the computer program IBM SPSS Statistics. Results: The AG genotype of IL-10 rs1800871 was statistically significantly lower in the inactive PA group than in the control group . The TG genotype of IL-10 rs1800872 was also statistically significantly lower in the inactive PA group than in the control group . A binary logistic regression analysis of the polymorphisms in the IL-10 gene in PA and control groups based on the pituitary adenoma activity showed that the AG genotype of IL-10 rs1800871 increased the chance of inactive PA by 2.2-fold in codominant  and overdominant  models. Moreover, the TG genotype of IL-10 rs1800872 increased the probability of inactive PA by 2.2-fold in codominant  and overdominant  models. The association of the IL-10 polymorphisms with PA invasiveness and recurrence in PA and control groups did not yield statistically significant results. Conclusions: IL-10 rs1800871 and IL-10 rs1800872 may be associated with the development of inactive PA.The aim and objective of this study is to determine the association between the rs1800871, rs1800872, and rs1800896 polymorphisms of the gene  Pituitary adenomas (PA) are described as benign monoclonal tumors in the pituitary gland that cause hormone hypersecretion or localization symptoms. PA are primary tumors that occur in the pituitary gland and are among the most common intracranial tumors, with a prevalence of approximately 0.1 percent in the population .The pathogenesis of pituitary adenomas is still poorly understood. Although many consider benign monoclonal proliferation, their clinical spectrum is diverse, including hormone hypersecretion and various degrees of invasiveness, which suggests multiple steps and mechanisms as well as molecular abnormalities in tumorigenesis and gene regulation of pituitary adenomas .IL-10 gene is located on chromosome 1q31–32 (10), a locus genetically associated with susceptibility to a number of autoimmune diseases : AGTGAGCAAACTGAGGCACAGAGAT[A/G]TTACATCACCTGTACAAGGGTACAC; rs1800872 Context Sequence [VIC/FAM]: CTTTCCAGACTGGCTTCCTACAG[T/G]ACAGGCGGTCACAGGATGTTC; and rs1800896 Context Sequence [VIC/FAM]: TCCTCTTACCTATCCCTACTTCCCC[T/C]TCCCAAAGAAGCCTTAGTAGTGTTG.Genotyping of PA invasiveness, activity, recurrence assessment, control group formation, DNA extraction, and genotyping have been described previously in our studies ,27.IL-10 serum levels were measured in 41 control subjects and 28 patients with PA. The assay was determined by enzyme-linked immunosorbent assay (ELISA) using the Invitrogen IL-10 Human ELISA Kit. This assay for human IL-10, with a standard curve sensibility range of 0.9–500 pg/mL and a sensitivity of <1 pg/mL was analyzed on the Multiskan FC Microplate Photometer  at 450 nm per the manufacturer’s instructions. The samples were excluded if the levels of serum cytokines were below the detection range.IL-10 gene rs1800871, rs1800872, and rs1800896 polymorphisms in the present report. Pearson’s χ2 statistical test was used in both the case and control subjects. The data were analyzed using SPSS 27.0 statistical analysis software . The frequencies of the IL-10 rs1800871, rs1800872, and rs1800896 genotypes and alleles between the case and control subjects were compared using the χ2 test in all groups. The nonparametric Mann–Whitney U test compared nonnormally distributed continuous data, and Pearson’s χ2 test was used to compare categorical variables. The genotype-based odds ratios (OR) and 95% confidence intervals (Cis) were estimated using binary logistic regression models regarding the effects of genotypes on the development of PA. The data are presented as absolute numbers with percentages in parentheses, a median, and interquartile range (IQR). A p-value of less than 0.05 indicated a statistically significant difference.We calculated the deviation from HWE of the IL-10 haplotype association analysis was performed separately in the patients with pituitary adenoma and control groups. We used the online SNPStats website (https://www.snpstats.net/snpstats/) (accessed on 12 March 2022). The pairwise linkage disequilibrium (LD) analysis was assessed by D’ and r2 measures. The associations between the haplotypes and multiple sclerosis were calculated by logistic regression and presented as ORs and 95% CI.The p = 0.069 and p = 0.199, respectively)  were women, and 46 (43.3%) were men. The subjects ranged from 18 to 84 years, with a mean age of 53.4 years. The control group consisted of 192 subjects, of whom 129 (67.2%) were women, and 63 (32.8%) were men. The age of the control group ranged from 18 to 94 years, with a mean age of 55.8 years. The sex and age did not differ between the groups (ctively) .We compared the distribution of genotypes and alleles’ frequencies between the PA and control groups and found no statistically significant differences . The binThe adenomas’ invasiveness distribution of the genotypes and alleles’ analysis was performed between the non-invasive PA group and the control group, as well as between the invasive PA group and the control group. However, the distribution analysis and binary logistic regression analysis did not reveal statistically significant results .p = 0.027), and the rs1800872 TG genotype was statistically significantly less frequent in the inactive PA group than in the control group   .IL-10 gene rs1800871, rs1800872, and rs1800896 on active/inactive PA development. The analysis revealed that IL-10 rs1800871 AG genotype increased the odds of inactive PA’s development 2.2.-fold under the codominant  and overdominant  models. Moreover, the IL-10 rs1800872 TG genotype increases the odds of inactive PA development 2.2-fold under the codominant  and overdominant  models  models .When comparing the distribution of the genotypes and alleles between the PA with the recurrence group vs. the control group and the PA without the recurrence group vs. the control group, no statistically significant differences were found ; the binp < 0.001)  vs. 8.474 (11.784), p = 0.016, accordingly). We noticed exactly the same results with rs1800872: the IL-10 levels were lower in the PA than in the control group with the GG and GT genotypes (median (IQR): 0.373 (1.023) vs. 0.924 (8.404), p = 0.018; 0.392 (1.665) vs. 8.474 (11.784), p = 0.016, accordingly). The IL-10 levels were also found to be statistically significantly lower in the PA than in the controls with the rs1800896 TT (median (IQR): 0.642 (1.634) vs. 7.796 (8.782), p = 0.007) and TC genotypes (median (IQR): 0.231 (4.430) vs. 8.300 (32.670), p = 0.010) : 0.328 (1.015) pg/mL vs. 5.9 (8.453) pg/mL, < 0.001) . A compa= 0.010) .Haplotype analysis was performed in the PA and the control groups. A pairwise linkage disequilibrium (LD) between rs1800871–rs1800872, rs1800871–rs1800896, and rs1800872–rs1800896 was observed  gene polymorphisms and IL-10 serum levels in patients with PA and healthy controls. The results were evaluated according to gender and the clinical course of the disease. To our knowledge, the associations of the IL-10 gene polymorphisms  and IL-10 serum levels with PA were analyzed for the first time.The study analyzed the associations of the IL-10’s gene transcription and translation, resulting in abnormal cell proliferation and cancer development [IL-10 is associated with many autoimmune diseases due to its anti-inflammatory functions. IL-10 inhibits innate and adaptive immune responses of leukocytes and limits potential tissue damage caused by inflammation. IL-10 is essential for host defense against various infections and developing many autoimmune diseases . Howeverelopment .IL-10 in association with PA. Huang and co-authors associated the gene expression of IL-10 with PA. The PRL (µg/L) levels were significantly higher, but the LH and FSH levels were lower in patients with non-invasive and invasive, non-functional PA compared to the control group. Furthermore, patients with invasive, non-functional PA had significantly lower white blood cell counts than patients with non-invasive, non-functional PA and the control group. The study showed that a high IL-10 expression in pituitary tumor tissues was associated with an increased invasiveness of non-functioning PA; thus, IL-10 levels in peripheral blood can be used as a diagnostic marker for invasive non-functioning PA [IL-10 rs1800871 and rs1800872 were associated with PA. The results of the study showed a statistically significant difference when comparing the inactive PA group with the control group. IL-10 rs1800871 AG and rs1800872 TG genotypes were less frequent in the inactive PA group than in the control group (p = 0.027). Depending on the activity of the PA, individuals with the IL-10 rs1800871 AG genotype or rs1800872 TG genotype were 2.2 times more likely to be an inactive PA in codominant and overdominant models. The other group of authors also studied patients with nonfunctioning but invasive PA (NFPAs) and found lower numbers of CD3-CD56 + natural killer (NK) cells and significantly higher levels of CD3 + CD8 + CD28 T cells (CD8 + Tregs) and interleukin-10 (IL-10) in peripheral blood. In addition, patients with invasive NFPAs had fewer infiltrated CD56 + cells, fewer infiltrated CD28 + cells, and significantly higher IL-10 expression. These results indicated that low infiltration of CD56 + cells and CD28 + cells and a high IL-10 expression in pituitary tumor tissues were related to an increased invasiveness of NFPAs. The levels of CD3-CD56 + NK cells, CD8 + Tregs, and IL-10 in peripheral blood may be useful as diagnostic markers for invasive NFPAs [Only one study has analyzed oning PA . In our ve NFPAs .IL-10 to other types of brain tumors, cancer, and CNS diseases. Several authors have linked the gene IL-10 to glioma cell proliferation. Zhang et al. link the IL-10 gene to glioma cell proliferation. The study showed that IL-10 increased cell growth and invasion into glioma cells. IL-10 has been shown to enhance the expression of KPNA2, which affects cell growth and expression in cancer cells, including the human brain tumor, suggesting that IL-10 promotes glioma cell growth and invasion by regulating KPNA2 [IL-10 gene is involved in the development of lymphoid malignancies. The study’s results showed that the level of IL-10 in the cerebrospinal fluid correlates positively with the tumor size and may help diagnose the development of lymphoid malignancies [IL-10 gene to primary and secondary CNS lymphomas. Concentrations of IL-10 in the cerebrospinal fluid in primary or secondary CNS lymphoma were also significantly higher than in samples from healthy subjects [Numerous studies have been conducted to link the gene ng KPNA2 . Anotherng KPNA2 . Geng angnancies . Lissonignancies . Rubenstsubjects .IL-10 as potential biomarkers in different types of cancer. Our study results showed that IL-10 rs1800871 and rs1800872 were associated with PA. Moreover, Wang and co-authors reported that the IL-10 rs1800871, rs1800872, and rs1800896 polymorphisms were significantly associated with the risk of gastric cancer in Asians under two models: dominant and additive. In this case, our study indicated that the IL-10 polymorphisms  could serve as genetic biomarkers of gastric cancer in Asians [IL-10 polymorphisms’ susceptibility to head and neck cancers (HNC). This study reported that the rs1800871 C/C genotype was associated with a 2.5-fold and 3.33-fold risk of developing nasopharyngeal cancer (NPC) and laryngeal cancer (LC), respectively, and carriers of the rs1800872 A/A genotype had a 2.5-fold increased risk of HNC and NPC, but not LC. The investigation findings showed that the IL-10 rs1800896, rs1800871, and rs1800872 SNPs contribute to the development of NPC, which suggests a possible role for these variants as biomarkers for the early detection of HNC and, in particular, the NPC subtype [IL-10 (rs1800896), the AA genotype and GA + AA genotype were associated with an enhanced risk of esophageal cancer; thus, this variant may be performed as a candidate biomarker for predicting the development of esophageal cancer [Numerous studies have investigated the association between the polymorphisms of n Asians . Moghimin Asians . Makni a subtype . In the l cancer .IL-9 (rs1859430) polymorphism are more prevalent among patients with a recurrent PA. Moreover, our results show the that A/A genotype and A allele are significantly more common among those patients for whom the PA did not reoccur. We can hypothesize that IL-9 may be important for PA recurrence, but it has no impact on hormonal activity or invasiveness [Our study found that the IL-10 levels were statistically significantly lower in the PA than in the control group with the rs1800871, rs1800872 GG and GA genotypes, and with the rs1800896 TT genotype. Several authors reported that IL-10 serum levels were important in cancer development. Bobe et al. revealed that elevated serum levels of IL-10 increased the risk of colorectal adenoma recurrence . Shokrzasiveness .IL-10 and the development of brain tumors, CNS alterations, and other types of cancers, it is possible that changes in gene expression influence disease.Based on the results of studies that investigated the association between the gene IL-10 gene in CNS diseases and brain tumors, especially PA. More research and an increase in sample size would provide more information on the effects of this gene on pituitary tumor development, progression, and activity.Currently, there are insufficient studies on the Personalized medicine plays the most important role in the prevention, diagnosis, prognosis, and therapy of neoplasms and cancer. It is important for clinical management in routine clinical practice and treatment. Identification of genes that are predisposed to neoplasms and cancer can help make decisions for an individual’s disease risk modification. The study of genes in different populations is very valuable because the prevalence of genes varies widely in very different ethnic groups. In the future, we need to focus on the role of personalized medicine, including the most important: one-gene therapy medicine.


The peptide was obtained with excellent purity and yield. We are confident that this strategy will be rapidly implemented by other peptide laboratories.Herein we report a practical approach for peptide synthesis using second-generation fibrous polyacrylamide resin . This resin with the corresponding handle was used for solid phase peptide synthesis (SPPS) using a fluorenylmethoxycarbonyl (Fmoc) approach. We reveal that the most appropriate mixing and filtration strategy when using amino-Li-resin in SPPS is via shaking and gravity filtration, instead of mechanical stirring and suction filtration used with other resins. The strategy was demonstrated with the SPPS of H-Tyr-Ile-Ile-Phe-Leu-NH Peptides are a key class of organic compounds. They are chemically synthesized by assembling two or more amino acids, which are joined by an amide bond formed from the interaction of the carboxylic end of one amino acid with the amino end of another ,2,3,4. VGiven the multidimensional use of peptides, efforts have been made over the years to optimize synthetic strategies for their preparation ,31,32,33The most critical aspect of SPPS is the resin, which must have particular features to be suitable for peptide synthesis. In this regard, the resin should have an enhanced capacity to hold the different reactive sites (loading > 0.5 mmol/g), which should be uniformly distributed to minimize interchain interactions and therefore facilitate elongation of the peptide chain. It should also have good swelling properties in the solvents used to carry out the different reactions, thus enabling access of the reagents to the reactive sites. Moreover, the resin should be mechanically stable and permit filtration for removing the excess of reagents and soluble side products.Polystyrene (PS)-based resins cross-linked with 1% of divinylbenzene are most commonly used in research laboratories and in large-scale peptide production by SPPS. Given that dichloromethane (DCM), which has been reported as hazardous, is the solvent of choice for swelling, the practicability of these resins is compromised. Similarly, PS-based solid support is further limited by its poor swelling capacity, particularly in polar solvents, which are the most adaptive for the synthesis of large peptides. In an attempt to change the paradigm (PS and DCM), significant efforts have been made to modify existing resins by increasing crosslinking or by adding new functionalities, thereby leading to new supports such as polyethylene glycol-PS (PEG-PS) ,40,49,50To overcome the poor swelling capacity of PS and uneven solvation of PEG in some solvents, which is associated with major resins in the market today, there is a need to find a solid-phase support that is more homogeneous in terms of swellability and more versatile in terms of compatibility with the reaction conditions commonly used for solid-phase synthesis. Recently, our group described the second generation of amino-polyacrylamide resin  for SPPS . This su2O and even in aqueous buffers. Moreover, amino-Li-resin has a high loading capacity and good chemical stability towards acidic and basic reagents. Here we describe a practical protocol for the use of the amino-Li-resin for SPPS using the synthesis of Ile2,Ile3-Leu-Enkephalin (H-YIIFL-NH2) as a model.An additional advantage of amino-Li-resin compared to PS and other resins is its excellent swelling capacity in HN,N’-dimethylformamide (DMF), acetonitrile (CH3CN), and methanol (MeOH), were purchased from Merck Pty, SA and used without further purification. The second-generation polyacrylamide resin (amino-Li-resin) with a 100–200 mesh particle size used herein was obtained from Biotide Core, LLC, Corvallis, OR, USA, and Fluorenylmethoxycarbonyl (Fmoc) amino acids were purchased from Iris Biotech GmbH . The coupling reagents N,N’-diisopropylcarbodiimide (DIC), OxymaPure®, and 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)] methanaminium hexafluorophosphate (COMU®) were a gift from Luxembourg Biotech, Israel. The rest of the reagents, such as piperidine (deprotecting agent), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA) (cleavage), and triisopropylsilane (TIS) , were also supplied by Merck Pty Ltd.  and used in this protocol as received.All procedures pertaining to the peptide synthesis were carried out in a polypropylene syringe fitted with a penetrable frit. The HPLC grade solvents used in this study, namely dichloromethane (DCM), 18 column  operating at 1.0 mL/min, with linear gradients of 0.1% TFA in MilliQ water and 0.1% TFA in CH3CN as eluents. Data processing was performed using the Chemstation software (B.02.01 SR1 version) . Similarly, Liquid Chromatography-Mass Spectrometry (LCMS) analysis was conducted on an Ultimate™ 3000, AerisTM  with a Phenomenex C18 column , a flow rate of 1.0 mL/min and UV detection of 220 nm, with a linear gradient of 0.1% formic acid in MilliQ water and 0.1% formic acid in CH3CN as eluents.All shakings were performed using a shaker operating at 120 rpm . Centrifugation was performed on a centrifuge . Peptide purity was determined by means of an Agilent 1100 HPLC system using a Phenomenex AerisTMC18  column, with a flow rate of 1.0 mL/min and UV detection at 220 nm. 10 µL of the pentapeptide was injected on a reversed-phase CAmino-Li-resin  was added to a 5 mL polypropylene syringe and subjected to swelling for 10 min in DMF (5 mL). The swelled resin was then pretreated with 5% DIEA in DCM (2 mL × 2) to neutralize the starting resin and then washed in DMF (2 mL × 2). A mixture of 5 eq. of Fmoc-Leu-OH , 5 eq. of DIC , and 5 eq. of OxymaPure  dissolved in DMF  was added to the washed resin and shaken for 3 h. The loaded resin was washed with DMF (3 mL × 2) and MeOH (3 mL × 2) and left to dry until constant weight. Next, 20 mg of the loaded resin was weighed and transferred to a polypropylene syringe fitted with a porous frit, and 20% piperidine in DMF (500 µL) was added and stirred for 10 min. With the aid of a piston, the filtrate was gently pushed through into a volumetric flask (25 mL) and rinsed further with DMF (3 mL × 3). The solution was then made up to the mark with DMF.−1 cm−1, obtained from the calibration curve of a freshly prepared Fmoc-Leu-OH solution. Using Equations (2) and (3), resin loading was then determined to be 0.4 mmol/g.Using a cuvette with a path length of 1 cm, the UV absorption profile for the solution was recorded using a UV spectrophotometer in the 270–320 nm range. The peak of the diagnostic dibenzofulvene adduct in the 298–301 nm range was used to determine the concentration of solution according to Equation (1), using a molar absorption coefficient L = Resin loading; c = concentration; v = volume of solution (volumetric flask); A = absorption maxima around 298–301 nm, l = path length of the cuvette.Amino-Li-resin  was added to a 20 mL polypropylene syringe fitted with a porous frit of 0.45 µm a. Next, In SPPS, resins are conventionally mixed by mechanical stirring, and filtration is performed using a vacuum source, both procedures tending to collapse the resin and clog the porous frit, thereby hampering the effectiveness of the entire process. To circumvent this deterioration, amino-Li-resin was gently mixed with the reagents by shaking  and filtAfter completion of the SPPS, images of the resin  using a 2O)  and 20 eq. of DIEA (1390.8 µL. 4 mmol) in DMF (3 mL) for 30 min. The solvent was then drained by gravity filtration and the resin was washed with DMF (5 mL × 3). Similar to what was established in Fmoc Rink-Amide linker was incorporated into the amino-Li-resin by reacting the swelled resin with 4 eq. of Fmoc Rink-Amide linker , 3.8 eq. COMU , a slightly lower amount of COMU was used to prevent the capping of the amino function with the formation of the corresponding guanidino derivative [Elongation of the sequence proceeded in a continuous cycle  beginninThe first amino acid was incorporated by the addition of a preactivated mixture (0.3 M) of 3 eq. Fmoc-Leu-OH  containing 3 eq. OxymaPure  and 3 eq. DIC  to the deprotected Rink-Amide-Li-resin, followed by gentle shaking for 1 h . The solAfter the final deprotection step in the last incorporation, the peptidyl resin was subjected to a final cleavage to detach the peptide. The cleavage cocktail solution was a freshly prepared mixture of TFA (12.35 mL), TIS (0.325 mL), and water (0.325 mL) in 95%:2.5%:2.5% ratio. The cocktail (1 mL per 100 mg of peptidyl resin) was added to a falcon tube containing the peptidyl resin and left to stir for 1 h. Given that TFA is highly corrosive, it should be used in a well-ventilated environment and discarded in an approved waste disposal plant.The peptide was precipitated after cleavage by the addition of 45–70 mL of cold diethyl ether  followed by intensive agitation, leading to the appearance of a whitish precipitate. The falcon tube was then centrifuged (3600 rev per min for 4 min). The supernatant layer was carefully decanted into a clean falcon tube and kept in ice for further precipitation of peptides that might have been released during decantation. The cleaved peptide was then washed twice with cold diethyl ether in the same manner and dried in vacuo to completely remove the ether. The peptide was finally extracted with 10% acetic acid (HOAc), then filtered under gravity, frozen with liquid nitrogen, and kept in a freeze dryer for 24–48 h until a powder was obtained.2,Ile3-Leu-Enkephalin (H-YIIFL-NH2) peptide after lyophilization (SR) was 0.2 mmol. Furthermore, according to Equation (5), the theoretical yield  had purity of 96.1%, thus confirming the suitability of the amino-Li-resin for SPPS.As shown in the chromatogram  obtained3, Ile4 -Leu Enkephalin using the Fmoc/tBu strategy via shaking and gravity filtration, which assures the integrity of this resin. Unlike the ChemMatrix resin, which has a very low yield, amino-Li-resin afforded a relatively high yield (81%) in the synthesis of the model peptide used in this protocol.Amino-Li-resin is a second-generation polyacrylamide support with a highly cross-linked matrix designed to confer the mechanical stability required for effective use in SPPS. It has excellent swelling capacity in a wide range of solvents, particularly in polar solvents such as water and alcohols, which are compatible with biochemical processes. The suitability of amino-Li-resin has been demonstrated in a stepwise synthesis of Ile